基于RNA-Seq测序的高、低产双黄蛋高邮鸭卵巢组织SNP筛选分析

doi:10.16431/j.cnki.1671-7236.2019.12.005

摘要/Abstract

摘要： 试验旨在进一步完善高邮鸭基因组结构信息，探索高邮鸭双黄蛋产蛋性能的调控机制。通过高通量测序技术对高邮鸭双黄蛋高、低产组卵巢组织转录组进行RNA-Seq测序，对测序数据进行质控和注释，筛选与高邮鸭双黄蛋产蛋性能相关的SNP位点。测序获得的初始数据（raw reads）进行系列质控筛选，6个高邮鸭卵巢组织分别获得84 540 140~87 479 660个有效数据（clean data），70.79%以上的测序数据被已注释的鸭基因组数据覆盖；在所覆盖的基因组数据中，检测到位于基因外显子区域的108 260~116 478个SNP位点（转换和颠换类型），转换类型总数极显著高于颠换类型总数（P<0.01）。利用KEGG数据库对筛选出的SNP位点所在基因进行信号通路分析，筛选出前20条信号通路，其中包括核糖体信号通路、碳代谢信号通路和氨基酸生物合成信号通路等。试验对双黄蛋高、低产组高邮鸭的卵巢进行了RNA-Seq测序分析，为发掘高邮鸭双黄蛋产蛋性能相关SNP位点提供了基础，为进一步研究高邮鸭双黄蛋产蛋性能的调控机制提供了新的数据支撑。

关键词: 转录组测序; 高邮鸭; 卵巢; 双黄蛋; SNP

Abstract: This study was aimed to improve the gene structure of Gaoyou duck,and explore its regulation mechanism of double-yolk egg performance.RNA-Seq technology was used to analyze the transcriptome of ovaries in high and low double-yolk egg yield Gaoyou ducks.SNP related to the double-yolk egg performance were screened by quality control and gene annotations from RNA-Seq data.Raw reads from RNA-Seq data were selected with quality control,84 540 140-87 479 660 clean reads were screened from total 6 samples,over 70.79% of the sequencing data were covered by the annotated genome data of ducks.Based on these data,108 260-116 478 SNP were found in the exon area of the annotched genes,and the number of transition SNP was extremely significant higher than the number of transversion(P<0.01).KEGG database was used to analyze the related pathways from these selected SNP.Top 20 pathways were screened,including Ribosome signaling pathway,Carbon metabolism signaling pathway,Biosynthesis of amino acids signaling pathway,and so on.The ovaries' transcriptome of Gaoyou ducks with high and low double-yolk performance were analyzed through RNA-Seq technology,the results provided the basis for mining and annotating new genes of Gaoyou ducks,and improved the data bases for the study of its regulation mechanism of double-yolk egg performance.

Key words: RNA-Seq; Gaoyou ducks; ovary; double-yolk egg; SNP

中图分类号:

S834

张蕾, 张海波, 章敬旗, 张扬, 常国斌, 陈国宏, 周丽君, 王健. 基于RNA-Seq测序的高、低产双黄蛋高邮鸭卵巢组织SNP筛选分析[J]. 中国畜牧兽医, 2019, 46(12): 3495-3503.

ZHANG Lei, ZHANG Haibo, ZHANG Jingqi, ZHANG Yang, CHANG Guobin, CHEN Guohong, ZHOU Lijun, WANG Jian. SNP Screening and Analysis of Ovaries Between Gaoyou Ducks with High and Low Yield Double-yolk Egg Based on RNA-Seq[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3495-3503.

参考文献

[1] MARIONI J C,MASON C E,MANE S M,et al.RNA-Seq:An assessment of technical reproducibility and comparison with gene expression arrays[J].Genome Research,2008,18(9):1509-1517.
[2] 李妍,徐兴祥.高通量测序技术的研究进展[J].中国医学工程,2019,27(3):32-37. LI Y,XU X X.Research progress of high-throughput sequencing technology[J].China Medical Engineering,2019,27(3):32-37.(in Chinese)
[3] LANDAU W M,LIU P.Dispersion estimation and its effect on test performance in RNA-Seq data analysis:A simulation-based comparison of methods[J].PLoS One,2013,8(12):e81415.
[4] MARIONI J C,MASON C E,MANE S M,et al.RNA-Seq:An assessment of technical reproducibility and comparison with gene expression arrays[J].Genome Research,2008,18(9):1509-1517.
[5] WILHELM B T,JOSETTE R L.RNA-Seq-Quantitative measurement of expression through massively parallel RNA-sequencing[J].Methods,2009,48(3):249-257.
[6] 崔凯,吴伟伟,刁其玉.转录组测序技术的研究和应用进展[J].生物技术通报,2019,35(7):181-190. CUI K,WU W W,DIAO Q Y.Application and research progress on transcriptomics[J].Biotechnology Bulletin,2019,35(7):181-190.(in Chinese)
[7] SPEEDY A W.Global production and consumption of animal source foods[J].Journal of Nutrition,2003,133(11):4048S-4053S.
[8] BOSCHIERO C,MOREIRA G C M,GHEYAS A A,et al.Genome-wide characterization of genetic variants and putative regions under selection in meat and egg-type chicken lines[J].BMC Genomics,2018,19(1):83-101.
[9] LIANG K,WANG X,TIAN X,et al.Molecular characterization and an 80-bp indel polymorphism within the prolactin receptor (PRLR) gene and its associations with chicken growth and carcass traits[J].3 Biotech,2019,9(8):296.
[10] 董新龙,张向前,韩昆鹏,等.基于RNA-Seq技术的京海黄鸡卵巢组织转录本预测及新基因挖掘[J].中国畜牧兽医,2017,44(2):497-504. DONG X L,ZHANG X Q,HAN K P,et al.Prediction of novel transcripts and discovery of novel gene on RNA-Seq analysis of Jinghai Yellow chicken ovary[J].China Animal Husbandry & Veterinary Medicine,2017,44(2):497-504.(in Chinese)
[11] 韩昆鹏,段炼,李婷婷,等.京海黄鸡卵巢转录组研究:基因结构分析与新基因发掘注释[J].中国畜牧兽医,2016,43(4):854-861. HAN K P,DUAN L,LI T T,et al.Study on RNA-Seq of Jinghai Yellow chicken ovary:Analysis of gene structure and mining new genes[J].China Animal Husbandry & Veterinary Medicine,2016,43(4):854-861.(in Chinese)
[12] 曹顶国.鸡繁殖性状相关基因功能分析及卵巢转录组和miRNAs鉴定[D].兰州:甘肃农业大学,2013. CAO D G.Functional analysis on reproduction-associated genes and identification of ovarian transcriptomes and mi RNAs in chicken[D].Lanzhou:Gansu Agricultural University,2013.(in Chinese)
[13] REN J,DU X,ZENG T,et al.Divergently expressed gene identification and interaction prediction of long noncoding RNA and mRNA involved in duck reproduction[J].Animal Reproduction Science,2017,185:8-17.
[14] 王坤,刘丽仙,谷大海,等.RNA-Seq在家禽中的应用研究进展[J].中国家禽,2017,39(15):50-56. WANG K,LIU L X,GU D H,et al.Research progress on application of RNA sequencing technology in poultry[J].China Poultry,2017,39(15):50-56.(in Chinese)
[15] 张蕾,张响英,章敬旗,等.高、低产双黄蛋高邮鸭卵巢组织差异分析[J].中国畜牧兽医,2018,45(7):1905-1911. ZHANG L,ZHANG X Y,ZHANG J Q,et al.Differential analysis of ovary structure between high and low double-yolk egg laying Gaoyou ducks[J].China Animal Husbandry & Veterinary Medicine,2018,45(7):1905-1911.(in Chinese)
[16] 陈杰,李慧芳,刘宏祥,等.高邮鸭与金定鸭GnRHR2基因组织表达差异分析[J].家畜生态学报,2011,32(2):13-18. CHEN J,LI H F,LIU H X,et al.Differential analysis of tissue expression of GnRHR2 mRNA in Gaoyou and Jingding duck breeds[J].Acta Ecologiae Animalis Domastici,2011,32(2):13-18.(in Chinese)
[17] 周根来,杨晓志,殷洁鑫,等.不同水平豌豆蛋白粉对高邮鸭生长及其相关激素与基因表达的影响[J].西南农业学报,2018,31(3):635-640. ZHOU G L,YANG X Z,YIN J X,et al.Effects of pea gluten meal on growth performance,serum hormones levels and expression of genes related to growth of Gaoyou duck aged from 0 to 4 weeks[J].Southwest China Journal of Agricultural Science,2018,31(3):635-640.(in Chinese)
[18] 郑蒙蒙,李伟,张蕾,等.高邮鸭产蛋性能研究进展[J].中国家禽,2017,39(17):19-51. ZHENG M M,LI W,ZHANG L,et al.Research progress on egg performance in Gaoyou duck[J].China Poultry,2017,39(17):19-51.(in Chinese)
[19] KIM D,PERTEA G,TRAPNELL C,et al.TopHat2:Accurate alignment of transcriptomes in the presence of insertions,deletions and gene fusions[J].Genome Biology,2013,14(4):R36.
[20] LANGMEAD B,SALZBERG S L.Fast gapped-read alignment with Bowtie 2[J].Nature Methods,2012,9(4):357-359.
[21] ETHERINGTON G J,RAMIREZ R H,MACLEAN D.Bio-samtools 2:A package for analysis and visualization of sequence and alignment data with SAMtools in Ruby[J].Bioinformatics,2015,31(15):2565-2567.
[22] SCHATZ M C,DELCHER A L,SALZBERG S L.Assembly of large genomes using second-generation sequencing[J].Genome Research,2010,20(9):1165-1173.
[23] HANSEN K D,STEVEN E B,SANDRINE D.Biases in Illumina transcriptome sequencing caused by random hexamer priming[J].Nucleic Acids Research,2010,38(12):e131.
[24] 李晓净.鸭蛋用性状的分子标记研究[D].武汉:华中农业大学,2017. LI X J.Study on molecular markers of egg traits in duck[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[25] 李慧芳,朱文奇,宋卫涛,等.催乳素(PRL)和生长激素(GH)基因对高邮鸭产蛋性能的遗传效应分析[J].农业生物技术学报,2009,17(2):263-268. LI H F,ZHU W Q,SONG W T,et al.Genetic effects of prolactin (PRL) and growth hormone (GH) genes on egg performance in Gaoyou duck[J].Journal of Agricultural Biotechnology,2009,17(2):263-268.(in Chinese)
[26] 朱文奇,李慧芳,宋卫涛,等.生长素基因Ghrelin对高邮鸭产蛋性能的遗传效应[J].四川农业大学学报,2009,27(3):354-360. ZHU W Q,LI H F,SONG W T,et al.The genetic effect of Ghrelin gene on egg performance in Gaoyou duck[J].Journal of Sichuan Agricultural University,2009,27(3):354-360.(in Chinese)